Low incidence of factor VIII inhibitors in previously untreated patients with severe haemophilia A treated with octanate® : Final report from a prospective study.

INTRODUCTION: Octanate® is a human, plasma-derived, von Willebrand factor-stabilized coagulation factor VIII (FVIII) concentrate with demonstrated haemostatic efficacy in previously treated patients with haemophilia A. AIM: This prospective, open-label study aimed to assess the immunogenicity of octanate® in previously untreated patients (PUPs). METHODS: The study monitored development of FVIII inhibitors in 51 PUPs. Tolerability, viral safety, FVIII recovery and efficacy of octanate® for the prevention and treatment of bleeds and in surgical procedures were also assessed. RESULTS: Five (9.8%) of the 51 patients developed inhibitors during the study, 4 of which (7.8%) were high titre. Three inhibitor cases (5.9%) were considered clinically relevant; 2 were transient inhibitors that disappeared during regular octanate® treatment without a change in dose or treatment frequency. Amongst 45 patients with FVIII:C <1% at baseline and who received ≥20 exposure days (EDs) or had <20 EDs but developed an inhibitor, inhibitor incidence was 11.1% (6.7% clinically relevant). All clinically relevant inhibitors developed within 20 EDs of on-demand treatment. No inhibitors developed in PUPs receiving prophylaxis. All patients who developed inhibitors had either intron 22 inversions or large deletions. Irrespective of the reason for administration, haemostatic efficacy was rated as "excellent" in 99.6% of all infusions (4700 of 4717 infusions), and no complications were reported in 23 surgical procedures. Mean incremental in vivo recovery was 2.0%/IU/kg (±0.7) and 1.9%/IU/kg (±0.5) for the first and second assessments, respectively. Tolerability was rated "very good" in 99.9% of infusions. CONCLUSION: In PUPs with severe haemophilia A, octanate® demonstrated haemostatic efficacy with a low rate of inhibitor development.

Immunogenicity, efficacy and safety of Nuwiq® (human-cl rhFVIII) in previously untreated patients with severe haemophilia A-Interim results from the NuProtect Study.

INTRODUCTION: Nuwiq® (Human-cl rhFVIII) is a fourth generation recombinant FVIII, produced in a human cell line, without chemical modification or protein fusion. No inhibitors developed in studies with Nuwiq® in 201 previously treated patients with haemophilia A (HA). The immunogenicity, efficacy and safety of Nuwiq® in previously untreated patients (PUPs) with severe HA are being assessed in the ongoing NuProtect study. METHODS: The study, conducted across 38 centres worldwide, is evaluating 110 true PUPs of all ages and ethnicities enrolled for study up to 100 exposure days (EDs) or 5 years maximum. The primary objective is to assess the immunogenicity of Nuwiq® (inhibitor activity ≥0.6 BU) using the Nijmegen-modified Bethesda assay at a central laboratory. RESULTS: Data for 66 PUPs with ≥20 EDs from a preplanned interim analysis were analysed. High-titre (HT) inhibitors developed in 8 of 66 patients after a median of 11.5 EDs (range 6-24). Five patients developed low-titre inhibitors (4 transient). The cumulative incidence (95% confidence interval) was 12.8% (4.5%, 21.2%) for HT inhibitors and 20.8% (10.7%, 31.0%) for all inhibitors. During inhibitor-free periods, median annualized bleeding rates during prophylaxis were 0 for spontaneous bleeds and 2.40 for all bleeds. Efficacy was rated as "excellent" or "good" in treating 91.8% of bleeds. Efficacy of surgical prophylaxis was "excellent" or "good" for 8 (89%) procedures and "moderate" for 1 (11%). No tolerability concerns were evident. CONCLUSION: These interim data show a cumulative incidence of 12.8% for HT inhibitors and convincing efficacy and tolerability in PUPs treated with Nuwiq® .

PK-guided personalized prophylaxis with Nuwiq® (human-cl rhFVIII) in adults with severe haemophilia A.

INTRODUCTION: Nuwiq® (human-cl rhFVIII) is a 4th generation recombinant human FVIII, without chemical modification or protein fusion, produced in a human cell-line. AIMS/METHODS: This study (NuPreviq) was a prospective, open-label, multicentre, phase IIIb study of the efficacy and safety of personalized prophylaxis with Nuwiq® in 66 previously treated adults with severe haemophilia A. NuPreviq had three phases: (i) a 72-h pharmacokinetic (PK) phase; (ii) a 1-3 month standard prophylaxis phase; and (iii) a 6-month personalized prophylaxis phase. The personalized prophylaxis regimen was based on individual PK modelling for each patient according to whether their PK profile most closely fitted a two- or one-compartment model (NuPreviq approach). In cases of uncertainty, a noncompartment model was applied. RESULTS: The median dosing interval during personalized prophylaxis was 3.5 days, with 57% of patients on ≤2 weekly dosing. Mean annualized bleeding rates during personalized prophylaxis were 1.45 (median [interquartile range, IQR]: 0 [0, 1.9]) for all bleeds, 0.79 (median [IQR]: 0 [0, 0]) for spontaneous bleeds, and 0.91 (median [IQR]: 0 [0, 0]) for joint bleeds. During personalized prophylaxis, 83.1% of patients were spontaneous bleed-free. Compared with standard prophylaxis, median weekly prophylaxis dose was reduced by 7.2% from 100.0 to 92.8 IU kg-1 during the last 2 months of personalized prophylaxis. There were no FVIII inhibitors or treatment-related serious or severe adverse events. CONCLUSION: PK-guided personalized prophylaxis with Nuwiq® provided bleeding protection and enabled the dosing interval to be extended to twice weekly or less in many patients and an overall dose reduction.

First prospective report on immune tolerance in poor risk haemophilia A inhibitor patients with a single factor VIII/von Willebrand factor concentrate in an observational immune tolerance induction study.

INTRODUCTION/BACKGROUND: Development of neutralizing inhibitors against factor VIII (FVIII) is a major complication of haemophilia A treatment. AIM: The ongoing, international, open-label, uncontrolled, observational immune tolerance induction (ObsITI) study evaluates ITI, the standard of care in patients with inhibitors. PATIENTS/METHODS: Forty-eight prospective patients in this interim analysis received a single plasma-derived, von Willebrand factor-stabilized, FVIII concentrate (pdFVIII/VWF) for ITI. According to recommended Bonn protocol, 'low responders' at ITI start (<5 BU) received 50-100 IU FVIII kg(-1) daily, or every other day; 'high responders' (≥5 BU) received 100 IU FVIII kg(-1) every 12 h. RESULTS: Forty of 48 patients (83.3%), had at least one risk factor for poor ITI-prognosis at ITI start (i.e. age ≥7 years, >2 years since inhibitor diagnosis, inhibitor titre ≥10 BU at the start of ITI, or prior ITI failure). Nonetheless, 34 patients (70.8%) achieved complete success, 3 (6.3%) partial success, 1 (2.1%) partial response; ITI failed in 10 patients (20.8%), all with poor prognosis factors. All six low responders achieved complete success. ITI outcome was significantly associated with inhibitor titre level at ITI start (P = 0.0068), number of poor prognosis factors for ITI success (P = 0.0187), monthly bleeding rate during ITI (P = 0.0005) and peak inhibitor titre during ITI (P = 0.0007). Twenty-two of 35 high responder patients (62.9%) with ≥1 poor prognosis factor achieved complete success. CONCLUSION: Treatment with a single pdFVIII/VWF concentrate, mainly according to the Bonn protocol, resulted in a high ITI success rate in haemophilia A patients with inhibitors and poor prognosis for ITI success.

Renaturation of rabbit liver aminoacyl-tRNA synthetases by 80S ribosomes.

Protein biosynthesis machinery is thought to be mostly compartmentalised within the mammalian cell, involving direct interactions between different components of the translation apparatus. The present research concerns the functional meaning of the interaction between the rabbit liver aminoacyl-tRNA synthetases and 80S ribosomes. We have shown that rabbit liver 80S ribosomes are able to enhance the activity of leucyl-tRNA synthetase, which is a component of high-molecular weight aminoacyl-tRNA synthetase complex, and phenylalanyl-tRNA synthetase not associated within this complex. The ribosomes increase the initial rate of both the total reaction of tRNA aminoacylation and the first step of this reaction, the formation of leucyladenylate. Moreover, a positive cooperativity of the tRNA interaction with two binding sites of leucyl-tRNA synthetase is also increased in the presence of highly purified 80S ribosomes. The effect of 80S ribosomes on partly denatured leucyl-tRNA synthetase and phenylalanyl-tRNA synthetase and the protection by 80S ribosomes of both enzymes against inactivation indicate a refolding and stabilising capacity of the ribosomes. It is concluded that the interaction of aminoacyl-tRNA synthetases and 80S ribosomes is important for the maintenance of an active conformation of the enzymes.

DeltaNp73 antisense activates PUMA and induces apoptosis in neuroblastoma cells.

The p73 gene codes for various different protein isoforms. They include proteins expressed under the control of the P1 promoter that contain a transactivation domain and are similar in function to p53 (TAp73 isoforms), as well as proteins regulated by the P2 promoter that lack this domain and function as dominant negative inhibitors of TAp73 and p53 (DeltaNp73 isoforms). Whereas TAp73 functions as a tumor suppressor with pro-apoptotic function, DeltaNp73 is likely to prevent the induction of apoptosis in tumor cells and to participate in oncogenesis. Here we used a loss-of-function strategy to assess the role of DeltaNp73 in SH-SY5Y neuroblastoma cells. An antisense oligonucleotide designed to target DeltaNp73 mRNA, but not TAp73, was used to effectively downregulate this transcript. DeltaNp73 downregulation was accompanied by increased levels of the pro-apoptotic BH3 family member PUMA at the mRNA and protein level, and by conformational activation of BAX which translocated to mitochondria. These DeltaNp73 antisense-mediated alterations led to the induction of apoptosis as detected by decreased cell viability, augmented DNA fragmentation and increased caspase-3 activity in cell lysates. Our results demonstrate the cytoprotective role of DeltaNp73 in neuroblastoma and suggest its use as a target for molecular intervention therapy.

Exposure on cell surface and extensive arginine methylation of ewing sarcoma (EWS) protein.

In contrast to the knowledge regarding the function of chimeric Ewing sarcoma (EWS) fusion proteins that arise from chromosomal translocation, the cellular function of the RNA binding EWS protein is poorly characterized. EWS protein had been found mainly in the nucleus. In this report we show that EWS protein is not only found in the nucleus and cytosol but also on cell surfaces. After cell-surface biotinylation, isoelectric focusing of membrane fraction, avidin-agarose extraction of biotinylated proteins, and SDS-polyacrylamide gel electrophoresis, EWS protein was identified by matrix-assisted laser desorption ionization and nanoelectrospray tandem mass spectrometry of in-gel-digested peptides. These analyses revealed that the protein, having repeated RGG motifs, is extensively asymmetrically dimethylated on arginine residues, the sites of which have been mapped by mass spectrometric methods. Out of a total of 30 Arg-Gly sequences, 29 arginines were found to be at least partially methylated. The Arg-Gly-Gly sequence was present in 21 of the 29 methylation sites, and in contrast to other methylated proteins, only 11 (38%) methylated arginine residues were found in the Gly-Arg-Gly sequence. The presence of Gly on the C-terminal side of the arginine residue seems to be a prerequisite for recognition by a protein-arginine N-methyltransferase (PRMT) catalyzing this asymmetric dimethylation reaction. One monomethylarginine and no symmetrically methylated arginine residue was found. The present findings imply that RNA-binding EWS protein shuttles from the nucleus to the cell surface in a methylated form, the role of which is discussed.